It is well known that the elevated intraocular pressure of primary open angle glaucoma is related to an increased resistance to the outflow of aqueous humor. The precise location of the resistance and the mechanisms by which aqueous humor outflow is impaired, however, are still not clear. Even in normal eyes, the locus of aqueous humor outflow resistance has not been pinpointed. In order to resolve these questions, novel imaging techniques are needed. Two-photon laser scanning microscopy is a promising imaging technique that has many advantages over conventional techniques, including higher scanning rate and lower phototoxicity. In addition, two-photon laser scanning microscopy has the significant advantage of being able to image structures deep within a tissue without incurring out-of-focus bleaching. Analyses from this microscopic technique are appropriate for observing the three-dimensional organization of organs. Its application, however, is limited to subjects that can be easily immobilized and apposed to a coverglass on the stage of a regular microscope. Attempts have recently been made to develop a miniaturized microendoscope. A two-photon endoscope system with a miniaturized probe has enabled real time imaging of various tissues, such as the brain, that sit inside a body cavity and/or are difficult to place on a regular microscope stage. Further improvements are required to use such endoscope systems for real time live cell/structure imaging of the outflow pathway in the eye. In particular, the microprobe/scanning system should be watertight, and, especially for ex vivo (ab interno) experiments, the probe should be mounted into the perfusion anterior segment culture in a watertight fashion. We propose to build a new two-photon endoscope that is specific for the eye and capable of studying live cell/structure of the outflow pathway. The system will be built with components including a femtosecond excitation laser, a laser delivery unit, a fluorescence detection unit, an imaging acquisition unit, and a new endoprobe/scanning system. Because understanding the outflow pathway necessitates real time imaging of live cell/structure, the two-photon endoscopy system will be used in an ex vivo perfusion human anterior segment culture as well as in in vivo studies of monkey eyes. To observe the live cell/structure of the human outflow pathway ab interno (from anterior chamber side), the two-photon endoscopic system will be mounted on an ex vivo perfusion human anterior segment culture system, and real time live cell/structure imaging will be established. For in vivo imaging of the aqueous outflow pathway, monkey eyes will be observed, and the aqueous outflow pathway will be observed ab externo (from outside of the eye) to image it without disturbing the anterior chamber. This will be attempted after scleral flaps have been removed, with and without removal of the outer wall of Schlemm's canal. Fluorescein staining for live tissue will be part of the experimental design. The findings of these research endeavors may lead to novel real time imaging methods for investigating the outflow pathway and will help illustrate the aqueous outflow route and events. PUBLIC HEALTH RELEVANCE: The elevation of intraocular pressure often associated with glaucoma is related to reduced drainage of the aqueous humor fluid from the eye. The drainage pathway of the aqueous outflow is difficult to view with current technology;therefore, a new imaging system, such as two-photon laser scanning microscopy, would provide a better understanding of this system which may lead to newer treatments for glaucoma. The goal of the project is to develop a new endoscope system that is specifically designed for the study of the eye with excellent resolution and scanning speed so that two-photon imaging of the living outflow pathway can be achieved.